Evaluation of the effect of Sm28GST-derived peptides in murine hepatosplenic schistosomiasis: Interest of the lipopeptidic form of the C-terminal peptide

MolecularImmunology,Vol. 31, No. 16, pp. 1247-1256, 19% Copyright 0 1994Elsevier Science Ltd Printed in Great Britain. All rights reserved 0161-5890(94)00103-O 0161-5890/94 $7.00 + 0.00

EVALUATION OF THE EFFECT OF Sm28GST-DERIVED PEPTIDES IN MURINE HEPATOSPLENIC SCHISTOSOMIASIS: INTEREST OF THE LIPOPEPTIDIC FORM OF THE C-TERMINAL PEPTIDE V. PANCRti,*[I I. WOLOWCZUK,* S. GUERRET,~ A. DELANOYE,*

M. BOSSUS,t H. GRAS-MASSE,t A. CAPRON and C. AURIAULT*

*Laboratoire d’Immunologie cellulaire de l’interface hgte/parasite et de la pathog&n&se parasitaire, CNRS URA 1854, Institut Pasteur, Lille, France; tLaboratoire de Chimie des biomol&ules, CNRS URA 1309, Institut Pasteur, Lille, France; SUnit de Physiopathologie cellulaire et mol~culaire des fibroses tissulaires, CNRS URA 1459, Institut Pasteur, Lyon, France; and §Laboratoire d’lmmunologie des maladies transmissibles et allergiques, INSERM U 167, Institut Pasteur, Lille, France (First received 23 March 1994; accepted in revised form 6 July 1994)

Abstract- Among the synthetic peptides derived from the 28-kDa Schistosoma mansoni glutathione S-transferase (Sm28GST), immunization with the C-terminal peptide comprising amino acid residues 190-211 induced a reduction in splenomegaly, in the number of hepatic eggs and in hepatic fibrosis in mice infected by Schistosoma mansoni. The absence of antibodies specific for the Sm28GST or for the 190-211 peptide observed in our conditions of immunization with this peptide argued in favour of the involvement of cellular-dependent mechanisms in the reduction in hepatic pathology. This was confirmed by the passive transfer of 190-211 peptide-specific T-cell enriched spleen cells which reproduced the protective effect conferred by immunization with the 190-211 peptide. These 190-211 peptide-specific cells produced little IL4 and high levels of IFN-y, a potent inhibitor of collagen synthesis. Furthermore, the use of a Iipopeptidic form of the 190-211 peptide significantly improved the reduction in hepatic pathology obtained with the uncoupled peptide and induced a durable protective response. These results provide encouraging information for the possible use of synthetic peptides in the immunoprophylaxis of Schistosomiasis. Key words: Schistosoma mansoni, peptide, lipopeptide,

INTRODUCTION ~an~5~i infection in mice and man commonly results in a long-term, stable disease state (Boros, 1989). In both species, the disease is associated with the continual, daily production of viable eggs by the intravascular worms. Many of the eggs do not reach the external environment and cause an inflammatory response when trapped in liver sinusoids, leading to largely cell-mediated granuloma formation and ultimately to hepatic fibrosis (Phillips and Lammie, 1986). This fibrosis involves the excessive deposition of newly synthesized connective tissue matrix, with collagen being the major component (Wyler et al., 1987). It is clear that this pathophysiologic scenario is carefully regulated by multiple subpopulations of distinct subsets of T lymphocytes (Chensue et al., 1981; Mathew and Boros, 1986; Perrin ~c~~~o~o~~

[IAuthor to whom correspondence should be addressed at: Laboratoire ~Immunolo~e cellulaire de l’interface h&e/ parasite et de la pathog&se parasitaire, CNRS URA 1854, Institut Pasteur, 1 rue du Pr. Calmette, B.P. 245, 59019 Lille, Ckdex, France.

liver pathology.

and Phillips, 1988; Fidel and Boros, 1990; Mathew et al., 1990; Grzych eb al., 1991; Pearce et al., 1991). Progress towards the development of a vaccine against schistosomiasis has been made both via our knowledge of the protective immune mechanisms and by the characterization and molecular cloning of target antigens. Among the most promising candidates, the 28-kDa S. mansoni glutathione S-transferase (Sm28GST) (Taylor et al., 1988) molecule has been the focus of several experimental studies allowing a better understanding of its immunogenicity and biological functions. The corresponding rSm28GST induced a significant degree of protection by immunization of permissive hosts such as hamsters, mice and baboons or of a semipermissive host such as the rat (Balloul et ai., 1987~; Wolowczuk et al., 1989) and also strongly reduced the egg granulomarelated pathology (Boulanger et al., 1991). We have recently demonstrated (V. Pancr6, in press) that i.p. administration of a single dose of rSm28GST, a protocol initially used to induce SmZZIGST-specific CD8+ CTL (V. Pan&, manuscript in preparation), also induced a reduction in hepatic pathology in infected mice. Both CD4+ and CD8+ T cell subpopulations were involved in

1247

1248

V. PANCR6

the expression of protection as demonstrated by the passive transfer of rSm28GST-specific T-cells. Moreover, we have shown that IFN-y, which is produced by Sm28GST-specific cells and is known to be a potent inhibitor of collagen synthesis (Czaja et al., 1987), could be responsible for the protective effect observed after immunization with rSm28GST. The possibility of using synthetic peptides as vaccines against viral, bacterial and parasitic diseases has evoked considerable interest during the last decade. At this stage of our study, it seemed interesting to determine which part of the Sm28GST molecule was the target of the response leading to the reduction of the lesions observed in the livers of infected mice. Previous peptide mapping of this molecule has identified a number of major epitopes. The 115-l 3 1 peptide contained both T- and B-cell recognition sites when tested in a variety of animals including rats, mice and monkeys (Auriault et al., 1991). Moreover, immunization with the octamerit (MAP 8) construction of the 115-l 3 I fragment has been shown to confer signi~cant protection to rats against a challenge infection in terms of a reduction of worm burden (Wolowczuk et al., 1991). A study using PBL from Kenyan patients infected with S. mansoni has identified the 190-211 peptide as one of the main T-cell epitopes (Wolowczuk et at., 1993). The 2443 peptide has been described as being predominantly involved in T-cell response in mice. In addition, immunization of mice with the lo--43 and 190-211 peptides induced a reduction in the numbers of intestinal and hepatic eggs and in their ability to hatch (Xu et al., 1993). In the present work, we tested the ability of Sm28GST-derived uncoupled peptides injected i.p. to protect S. mansoniinfected mice, The effect was evaluated in terms of the reduction in splenomegaly and hepatic lesions and we demonstrated that in these conditions the 190-211 peptide is able to reproduce the protective effect of the entire molecule. Furthermore, the use of a lipopeptidic form of the 190-211 peptide significantly improved the protection obtained with the uncoupled peptide. MATERIALS AND METHODS Animals

Female BALB/c mice, 68 weeks old at the beginning of the experiments, were provided by Iffa Credo (L’Arbresle, France). Parasite life cycle

A Puerto-Rican strain of S. mansoni was maintained in Biomphalaria glabrata snails as intermediate hosts and golden hamsters as definitive hosts. Cercariae for experimental infections were used within 1 hr after collection and enumeration, from l-month-infected snails exposed to light and to a temperature of 30°C for 1 hr. Recombinant Sm28GST

antigen and synthetic peptides

A full length cDNA encoding for the Sm28GST of S. mansoni was expressed in Escherichia coli using a PL-

et al

expression vector (Courtney et al.. 1984). The purified recombinant Sm28GST protein (rSm28GST) (95% pure after passage throught a glutathione-Sepharose column) was provided by F. Trottein from the Tnstitut Pasteur (Lille, France). Eight peptides were synthesized by solid phase methodology (Merrifield, 1983) derived from the Sm28GST (Balloul et al., 19876) amino acid sequence published in the Protein Sequence Data Bank (National Biomedical Research Foundation, Georgetown University Medical Center, Georgetown, DC) as the following: residues 1O-36, YFDGRGRAESIRMTLVAAGVDYEDERI; 2443, LVAAGVDYEDERTSFQDWPK; 65-86, VKWMLESLAIARYMAKKHHMMG; 87-I 11, ETDEEYYSVEKLIGQAEDVEHEYHK~ 11% 13t , KPQEEKEKITKETLNGK; 140-153, CESLKGSTGKLAVG; 1588175, LADLVLIAVIDHVTTDLDK; 190-211, GNLLASSPRLAKYLSNRPATFP. Multiple antigenic preparation of rhe 190-211 (MAP-I))

peptide

The synthesis of chloroacetylated core matrix, ClCH,CO-Map-Gly-CONH,, was accomplished manually as described (Lu et al., 1991) by a stepwise solid phase procedure on a BOC-Gly-paramethylbenzydrylamine (MBA) resin with a load of 0.1 mmol/g. After the removal of the BOC group by 50% tri~uoroacetic acid and neutralization of the resulting salt by 5% diisopropylethylamine (DIEA), the synthesis of three levels of the core matrix to form the octabranching BOC-Lys,Lys,-Lys-Gly-MBHA resin (Tam, 1988) was achieved using a 2.5 M excess of N-‘BOC, N-‘BOC-L-lysine dicyclohexylamine salt and benzotriazol- I-yloxy-Tris(dimethylamino)-phosphonium hexafluoro-phosphate (BOP) (Castro et al., 1975) as a coupling agent. After the removal of the last BOC group and neutralization, chloroacetic acid was coupled to the core matrix using symmetric anhydride activation in dichloromethane. The CICH*CO-MAP-Gly-MBHA resin was cleaved by the high hydrogen fluoride (HF) method, and the product was purified by reversed-phase HPLC and checked by analytical HPLC, mass spectometry and amino acid analysis. Five miliiliters of deionized water containing 2.8 g of guanidine hydrochloride and 13 mg of EDTA were added to 40 mg (16 pmol) of peptide 190-211 and DTT (3 mg) in a screw cap vial maintained under nitrogen. The solution was adjusted to pH 6.4-6.5 by 1 M Tris-HCl, pH 8.7 and flushed with N, for 30min, then left capped for 12 hr. Core matrix (2.3 mg) was added, the pH was adjusted to 8.7 and the solution stirred for 45 mn. This solution was exhaustively dialysed against 100 mM NH,HCO, and lyophilized in 20 ml 0.1 M phosphate buffer. Lipo 190-211 peptide

~-Aminohexadecanoic acid (Hda) was obtained by nucleophilic substitution of cr-bromohexadecanoic acid by ammonia. The N-rx-tertiobutyl-oxycarbonyl group was introduced by action of the tertiobutyldicarbonate on the Hda. After the introduction of the last amino

Protective effect of Sm28GST-derived C-terminal peptide

1249

acid, the N-terminal group of the 190-211 peptide was deprotected using 50% trifluoroacetic acid, neutralized, and the Hda residue was introduced using BOP as a coupling agent. Final deprotection and cleavage of the peptidyl resin were performed by the HF procedure for I hr at 0°C. The cleaved deprotected peptide was precipitated with cold diethylether and then dissolved in 10% acetic acid and lyophilized. Crude peptide was purified by reversed-phase HPLC and checked by analytical HPLC, mass spectometry and amino acid analysis.

were then washed twice in PBS containing 0.3% BSA before the addition of 0.1 ml of appropriately diluted mouse sera. The plates were then incubated at room temperature for 24 hr, then again washed twice in PBS-O.3% BSA. A final 18-hr incubation with 10’ cpm ‘251-1abe1edanti-mouse IgA Ab (Sigma) was effected to detect the presence of IgA specific for Sm28GST in the sera. After washing, as described above, the wells were cut out and counted in a gamma counter (LKB, Broma, Sweden) with a counting efficiency of 60%.

Immunization procedure

Lymphocyte culture medium

Mice were injected intraperitoneally with 50 pg of rSm28GST or peptides mixed with alumunium hydroxide (AH, Serva, Heildelberg, Germany). On days 14 and 21 peptide-immunized animals were reinjected with peptides (25 pg/mouse) in AH using the same route. Control mice were injected with saline buffer or with the irrelevant peptide 231-250 of the Toxoplasma gondii P24 antigen, using the same protocol.

For all cultures, RPM1 1640 (Gibco, Courbevoie, France) was supplemented with 5 x 10m5M p-mercaptoethanol (Merck, Darmstadt, Germany), 2 mM L-glutamine (Merck), 1 mM sodium pyruvate (Gibco), antibiotics (100 IU/ml penicillin, 100 fig/ml gentamycin) (Specia, Paris, France), 20mM HEPES (Sigma) and 10% heat inactivated foetal calf serum (Gibco) (ML 10). Lymphocyte proliferation assay

Infection protocols

Animals were exposed percutaneously to S. mansoni cercariae 1 day after the single (for rSm28GST) or last (for peptides) injection. Mice in the different groups were bled weekly through the retro-orbital sinus and finally sacrificed. Livers and spleens were removed, measured, weighed and fixed in Bouin liquor. ELZSA

Microtiter plates (Dynatech, Denkendorf, Germany) were incubated overnight at 4°C with 5 pg/ml of rSm28GST or peptides in 0.1 ml of sodium carbonate buffer (15 mM Na,CO,, 35 mM NaHCO,, pH 9.6). The plates were then saturated for 1 hr with PBS-BSA 3%. After careful washes, 0.1 ml of mouse serum diluted 1/SO in dilution buffer (0.15 M NaCl, 0.05% Tween 20, 10 mM PBS, pH 7.2) was dispensed into each well and incubated overnight at 4°C. After additional washes, 0.1 ml peroxidase labeled anti-mouse IgG (Diagnostic Pasteur, Marnes-la-Coquette, France) was added at a dilution of l/5000 in the same buffer for 1 hr at room temperature. After a final wash, 10 mg/ml substrate (Orthophenyldiamine, Sigma, St Louis, MO) in sodium phosphate buffer (0.1 M, pH 5.5), containing H,O, (1 ml/l) was incubated for 15 min at room temperature and the reaction was stopped by the addition of 50 ~1 of HCl(1 N). The O.D. was measured using a multichannel spectrophotometer (Titertek Multiskan MCC 1340) at 492 nm. Results are expressed as the mean of duplicate wells after subtraction of the background. Radioimmunoassay

Microtiter plate wells (microtest flexible assay plate, Falcon, Oxnard, CA) were coated for 24 hr at room temperature with 0.1 ml/l pg of rSm28GST or peptides in Na,CO, buffer (0.1 M, pH 9.6). A 2-hr saturation at room temperature was then carried out by the addition of 0.1 ml PBS (0.1 M) containing 3% BSA. The plates

Spleens cells were isolated from day 21-190-211 peptide-immunized mice, enriched in T-cell populations by passage through a Nylon wool column and maintained at 37°C in a 5% CO, atmosphere in lymphocyte culture medium. For the in vitro assays, 5 x 10’ T-cells and lo6 syngeneic irradiated splenic cells as APC were cultured with rSm28GST (10-80 pg/ml) or BSA (50 pg/ml) in a total volume of 0.2 ml in flat-bottom microtiter tissue culture plates (Nunclon, Roskilde, Denmark). The cells were then exposed to 18.5 kBq of tritiated deoxythymidine (3H-TdR) for the last 18 hr of a 5-day culture period. Finally, the cells were harvested by filtration on fiberglass discs using a multiharvester (Skatron, Lierbyen, Norway) and the amount of incorporated ‘H-TdR was measured using a liquid scintillation counter (LKB, Wallac, Turku, Finland). Data are expressed as the arithmetic mean of counts per minute (cpm) of triplicate cultures. Lymphokine production

Spleen cells were isolated from day 21-19&211 peptide-immunized mice and lymphokines were obtained by in vitro stimulation of 5 x IO6 cells with 40 pg/ml rSm28GST. Supernatant fluids were aliquoted and frozen after collection at 24 and 48 hr for determination of IL2 and IL4 and after 60 and 72 hr for determination of IFN-y. Lymphokine assays

Culture supernatants were assayed for IL2 by using the CTLL-20 cell line (ATCC, Rockville, MD). The supernatants were serially diluted in culture medium and plated in triplicate in 96-well round-bottom plates (Nunc). CTLL cells, maintained in ML10 supplemented with 5% Con A-stimulated mouse spleen supernatant, were cultured in a volume of 200 ~1 per well. After I8 to 20 hr of culture and a 4 to 6 hr pulse with I IlCi of 3H-thymidine, incorporation was measured. Test

V. PANCRk et al.

1250

supernatants were compared with a standard curve generated with rIL2 (Genzyme, Boston, MA). IL4 was measured using the ELISA method previously described. Briefly, plates were coated 3 hr at 37°C with 1 pg/ml rat anti-murine IL4 mAb (PharMingen, San Diego, CA) in 0.1 ml of sodium carbonate buffer and then washed with PBS-l% Tween. After a blocking step of 2 hr with 1% BSA, serial dilutions of test supernatants in a volume of 0.1 ml were added to the wells and incubated overnight at 4°C. After washing, 1 pg/ml biotin-labeled rat anti-murine IL4 Ab (PharMingen) was added for 2 hr at 37°C. After additional washes, peroxidase labeled Streptavidin conjugate (PharMingen) was added at a dilution of l/l000 for 1 hr at room temperature, followed by washing and substrate addition. Test supernatant values were compared with a standard curve generated with rIL4 (Genzyme). Culture supernatants were assayed for IFN-y with an ELISA kit (Genzyme) that utilizes a hamster anti-mouse IFN-y epitope-specific mAb and a goat anti-mouse IFN-y polyclonal Ab with multiple epitope specificity. A standard curve was generated by serial dilutions of a 8200 pg/ml solution of rIFN-y. The kit accurately measures IFN-y between 50 and 8200 pg/ml. Adoptive

Sirius red at 540 nm. Values were obtained with the use of the formula as previously described (Lopez de Leon & Rojkind, 1985). Student’s t-test and the coefficient of correlation were used in the statistical evaluation of the results. Data are reported as mean ? S.E.

RESULTS

Eflect qf immunization on splenomegaly

It?Zl rSm

T-cell enriched spleen cells (15 x 106) specific for the 190-211 peptide were injected iv. in 0.2 ml PBS into normal mice 18 hr before exposure to S. mansoni cercariae.

4

28CST

I

IO-36

tZ

24-43

0

65-86

E

87-111

c9

115-131

a

140-153

I

158-175

La

190-211

number determination

Liver sections, embedded in paraffin and stained using hematoxylin/eosin, were examined using a Leitz Asm 68 K image analyser (Wild leitz, Rueil-Malmaison, France) connected to a Leitz Diaplan microscope. All the granulomas present on the section were counted and the surface of the section was measured in order to determine the mean number for a defined surface. Significant differences (% reduction) in granuloma number were calculated between different groups and control mice using Student’s t-test. Measurement

peptides

Figure 1 shows the modifications in the size and weight of spleens of mice immunized with rSm28GSTderived peptides during S. mansoni infection. Mice immunized with rSm28GST or with the 190-211 peptide showed a significant reduction in S. mansoni-induced splenomegaly not observed after immunization with the other rSm28GST-derived peptides or with the T. gondii P2Cderived peptide as an irrelevant control (data not shown).

transfer experiments

Granuloma

with rSm28GST-derived

0.8

D 42

D 69

D 42

D 69

r

of collagen

The method was applied to 4 pm-thick sections of liver that were placed on slides, deparaffinized and incubated with a saturated solution of picric acid in distilled water containing 0.1% Fast Green FCF (Sigma) which stains noncollagenous proteins, and 0.1% Sirius red F3B (Gurr BDH Chemicals Ltd, Poole, U.K.) which stains collagen. Sections were kept out of the light and incubated at room temperature for 2 hr. They were then rinsed with distilled water until the elution fluid was completely free of color. Each slide was then covered with 1 ml of 0.1 N NaOH in absolute methanol (1: 1, v:v) and kept until all the color was eluted from the section (usually within a few seconds). Fluids were carefully withdrawn and read in a DU 64 spectrophotometer (Beckman Instruments Inc., Fullerton, CA). Fast green has its maximal absorbance at 630 nm and

Fig. 1. Levels of splenomegaly after immunization of mice with rSm28GST or derived peptides. Immunizations were performed i.p. with 5Opg of rSm28GST or peptides in the presence of AH. Control mice received adjuvant alone. On days 14 and 21, peptide-immunized mice were reinjected with peptides (25 pg) in AH using the same route. Mice were challenged with 50 cercariae, 1 day after the single (for rSm28GST) or last (for peptides) injection and sacrified at different times of S. mansoni infection. The sizes and weights of spleens were then evaluated.

Protective effect of Sm28GST-derived Table 1. Numbers of hepatic eggs after immunization with rSm28GST-derived peptides

of mice

Nbr hepatic eggs (% reduction) D 69 D 42 AH Sm28GST l&36 24-43 65-86 87-l 11 115-131 140-153 158-175 190-211 hr. Pept.

67 + 05 32 & 10 (52%) 60 + 07 (10%) 62 f 10 (7%) 66 f 05 (10%) 59f lO(ll%) 59 & 05 (11%) 51 k 05 (23%) 58 + 10(13%) 38 f 05 (43%) 61 + 10 (9%)

211+22 112f 12(46%) 198 f 18 (6%) 218 + 25 (0%) 205 & 10 (2%) 220 f 15 (0%) 190 + 12 (10%) 170 f 10 (19%) 192 k 25 (9%) 136 k 18 (35%) 180 k 30 (15%)

were performed i.p. with 50 pg of rSm28GST or peptides in the presence of AH. Control mice received adjuvant alone. On days 14 and 21 animals were reinjected

Immunizations

with peptides (25 pg) in AH using the same route. Mice were challenged with 50 cercariae 1 day after the single (for rSm28GST) or the last (for peptides) injection. Analyses were performed on defined liver sections (n = 3/mouse) sampled at day 42 and 69 of infection and the number of eggs present in each section was determined. The protection was expressed as the percent reduction of the mean hepatic egg number in immunized mice (n = 10) compared to adjuvant control mice (n = 10). Effect of immunization with rSm 28GST-derived peptides on hepatic pathology

The effect of immunization with rSm28GST-derived peptides on hepatic pathology was quantified by two different parameters: Efect on the number of hepatic eggs. The study was performed on liver sections sampled at days 42 and 69 after infection by S. mansoni (Table 1). A marked reduction in the number of hepatic eggs was observed, in particular at day 42 of infection, in mice immunized with the 190-211 peptide (43%, p < 0.05) or with rSm28GST, confirming previously described results (V. Pancre, in press). No significant reductions were seen in mice immunized with the other peptides in comparison with control animals immunized with the irrelevant peptide or AH alone. Efict on hepaticfibrosis. Fibrosis was quantified by the measurement of total collagen and protein content in liver sections of infected animals (Table 2). Results were expressed after subtraction of collagen content/mg protein in normal mice 42 or 69 days after the beginning of the experiment. We observed a significant diminution in collagen content/mg protein in mice immunized with 19&211 peptide ranging from 40 to 32% respectively, at days 42 and 69 after infection when compared with the infection control group. Thus after administration of the 190-211 peptide, the level of collagen in the liver was close to that observed in normal mice. Immunization with this peptide not only reduced hepatic pathology but also durably protected the mice, since the immunized animals survived to the infection MlMM 3,11&-D

1251

C-terminal peptide

and were sacrificed, whereas the control mice died after about 100 days of infection. The mice immunized with the other peptides died rapidly at about 75 days after infection with 10-36, 2443, 65-86, 87-l 11 peptides and around 100 days after infection with 115-131, 140-153 and 158-l 75 peptides, as was the case for control mice (data not shown). Thus, among the Sm28GST-derived synthetic peptides tested, the 19&211 peptide seemed to be of particular interest for a follow-up study. Antibody response after 190-211 peptide administration

Ab production was investigated in mice immunized with the 190-211 peptide. In our experimental conditions, we observed no detectable production of IgA or IgG Ab specific for the 19&211 peptide or for the Sm28GST molecule. During infection by S. mansoni, we obtained no significant difference in the Ab response against the Sm28GST molecule (Fig. 2) or against schistosomulum and egg antigens (data not shown) between peptide 190-21 l-immunized mice and the control groups immunized with adjuvant alone or with the irrelevant peptide. Cellular response to the 19&211 peptide

T-cell-enriched spleen cells from 19&211 peptideimmunized mice exhibited a significant, dose-dependent and highly reproducible proliferative response after incubation in vitro with rSm28GST (Fig. 3). In contrast, no stimulation was observed with BSA used as control Ag.

Table 2. Evaluation of hepatic fibrosis after immunization of mice with rSm28GST-derived peptides pg collagen/mg protein (% reduction) D 42 D69 AH Sm28GST lo-36 24-43 65-86 87-111 115-131 140-I 53 158-175 190-211 Irr. Pept.

8.4 f 0.9 3.8 &-0.4 (54%) 7.7 k 0.5 (8%) 8.2 k 0.5 (2%) 8.2 +_1.4(2%) 8.3 f 1.6(1%) 7.7 + 0.9 (8%) 7.2 &-0.5 (14%) 7.7 f 0.5 (8%) 5.1 + 0.5 (40%) 7.8 f 0.6 (7%)

13.3* 1.4 7.7 + 0.5 (42%) 13.0* 0.5 (2%) 13.5f 1.5(0%) 12.8+ 2.4 (4%) 13.5f 2.0 (0%) 12.7f 1.6(4%) 12.0f 0.5 (10%) 13.0f 1.6(2%) 9.1 f 1.3(32%) 12.8+ 1.5(4%)

Immunizations were performed i.p. with 50 pg of rSm28GST or peptides in the presence of AH. Control mice received adjuvant alone. On days 14 and 21 animals were reinjected with peptides (25 pg) in AH using the same route. Mice were challenged with 50 cercariae 1 day after the single (for rSm28GST) or last (for peptides) injection. The hepatic fibrosis was quantified by the measurement of collagen and protein content in liver section (n = 3/mouse) sampled at day 42 and 69 of infection by S. mansoni. Results were expressed after subtraction of collagen content/mg protein obtained in normal mice 42 and 69 days after the beginning of the experiment (respectively, 40.2 f 1.4, and 41.2 +_2.1). The protection was expressed as the percent reduction of the mean collagen/protein content in immunized mice (n = 10) compared to adjuvant control mice (n = 10).

1252

V. PANCR6

et al.

0.6

-

3 2

(A)

2 -

E

2

0 24 hr

48 hr

24 hr

Medium

48 hr

rSm28GST

3 DO

D28

D42

D62

D96

(RI

8000

6000

I

0

_

DO

D14

D28

D42

D62

D96

Fig. 2. IgG and IgA Ab profiles against Sm28GST in 190-211 peptide (lE#), irrelevant peptide (El) or AH alone (lJ)-immunized mice during the experimental course of S. mansoni infection.

Spleen cells from 19&211 peptide-immunized mice stimulated with 40pgg/ml of rSm28GST were tested for their capacity to produce IL2, IL4 and TFN-y (Fig. 4). After stimulation, we observed no significant production of IL4 (A), a weak production of IL2 (B) and an elevated level of production of IFN-y ranging from 500 to 800 pg/ml (C). During infection, the level of IFN-y obtained in 190-211 peptide-immunized mice remained superior to that observed in control mice injected with

z ,”

2000

24 hr

48 hr

rSm28GST

800

-s > a g

600 400

5 200

60 hr

12 Ix

Medium

60 hr

12 hr

rSm28GST

Fig. 4. Production of IL4 (A), IL2 (B) and IFN-1, (C) by spleen cells isolated from 190-2 11 peptide-immunized mice. Cells were stimulated with 40pg/ml of rSm28GST. The results shown are the means and SE of three experiments with five mice in each experiment.

AH alone (Fig. 5). This raised the question of the role of the IFN-y synthesized during infection by 190-211 peptide-specific cells. Passive transfer spectjic T cells

3000

4X hr

Medium

_

1000

I

I 24 hr

of immunity

with

190-211

peptide-

The protective effect of 190-211 peptide-specific T-cell enriched spleen cells on hepatic pathology was evaluated after their injection into syngeneic recipients 1 day before

-

2 a 5 e!

800 ,-IO00

? ,”

2

600

E -Gil ,a 400

0 APC

T

T

IO

20

40

80

BSA

+APC

% 5

200

Sm28GST

Fig. 3. In vitro proliferative response of T lymphocytes from 190-211 peptide-immunized mice after stimulation with rSm28GST (from 10 to 80 pg/ml). Results are expressed as the means and SE of three experiments in triplicate. T, APC and T+ APC represent the cpm obtained after in vitro incubation of lymphocytes alone, APC alone and lymphocytes with APC, respectively. It corresponds to the background proliferation of the cells, one of our negative controls (the other being the proliferative response obtained with BSA (50gg/ml) as irrelevant Ag).

0 NS

Sm28GST

Fig. 5. Production of IFN-y by spleen cells isolated from 190-211 peptide-immunized mice infected by S. mansoni. Peptide-immunized mice ( n) or control mice injected with AH alone (0) were challenged with 50 cercariae 1 day after the last injection. IFN-y production was evaluated after 72 hr stimulation with 40 pg/ml of rSm28GST. The results shown are the means and SE of three experiments with five mice in each experiment.

Protective effect of Sm28GST-derived

1253

C-terminal peptide

Table 3. Reduction of hepatic pathology after passive transfer of T-cell enriched spleen cells from 190-211 peptide-immunized mice Nbr hepatic eggs (% reduction) D 96 D 42 D 69 T-cells

65 f 07 45 f 05 (30%)

158 f 21 111* 11 (29%)

271 f 12 212f07 (21%)

pg collagen/mg protein (% reduction) D 96 D 69 D 42 8.2 f 0.5 5.7 f 0.2 (30%)

15.5 + 1.4 10.7 + 1.8 (30%)

23.5 rt 1.4 18.8 + 0.2 (20%)

T-cell enriched spleen cells from 190-211 peptide-immunized mice were passively transferred to syngenic mice 1 day before S. mansoni infection. Analysis was performed on defined liver sections (n = 3/mouse) sampled at day 42, 69, 96 of infection. The number of eggs present in the section were determined. Hepatic fibrosis was quantified by the measurement of collagen and protein content in the section and results were expressed after subtraction of collagen content/mg protein obtained in normal mice 42, 69 or 96 days after the beginning of the experiment (respectively, 40.2 f 1.4; 41.2 k 1.4 and 41.8 f 2.1). The protection was expressed as the percent reduction of the mean of the result in transferred mice (n = 10) compared to control mice (n = 10) that received only medium.

S. mansoni infection. As seen in Table 3, the transfer of the total T-cell enriched population led to reduction of about 30% in both the number of hepatic eggs and collagen content when the protective effect was maximal. This demonstrated that 190-211 peptide-specific cells reproduced the protective effect conferred by immunization with the peptide. Effect of the octameric (MAP-8) and lipopeptidic forms of peptide 190-211 on hepatic pathology

The protective effect of 190-211 peptide on hepatic lesions was maximal at day 42 of infection. In the majority of the experiments performed, this effect strongly diminished at day 96 of infection despite the fact that the mice survived, in contrast to the control group (Table 4). We tested the capacity of two different forms of the peptide 190-211, the lipopeptide 190-211

and the octameric construction of peptide 190-211, to induce a persistent reduction of the hepatic lesions observed on liver sections sampled at days 42,69 and 96 after infection by S. mansoni. E&ct on the number of hepatic eggs. The reduction in the number of hepatic eggs observed after administration of 190-211 peptide was even more marked when the peptide was presented in the lipopeptidic form (Table 4). Under these conditions, a significant protective effect was also observed up to 96 days after infection. In contrast, immunization with the octameric form induced no modification in the granuloma number in comparison to that obtained with the 19&211 peptide monomer. E&ct on hepatic fibrosis. The administration of the lipopeptidic form also induced a marked and stable reduction in the collagen content in the liver whereas no modification was obtained in mice immunized with the

Table 4. Evaluation of hepatic pathology after immunization of mice with the lipopeptidic and MAP-8 forms of 190-211 peptide Nbr hepatic eggs (% reduction) D 42 D 69 D 96 AH 190-211 Lip0 MAP-8

67_+05 (-) 38 + 05 (43%) 27 f 05 (49%) 41 + 10 (39%)

211 f21 (-) 136 + 18 (35%) 114f 18 (45%) 148 f 05 (29%)

294 * 35 (-) 248 + 12 (15%) 215 + 10 (27%) 262 f 17 (10%)

pg collagen/mg protein (X reduction) D 42 D 69 D 96 8.4 f 0.9 (-) 5.1 f 0.5 (40%) 4.9 + 0.4 (42%) 5.6 f 0.5 (33%)

13.3 f 1.4 (-) 9.1 + 1.3 (32%) 8.2 f 0.6 (38%) 9.4 f 0.6 (29%)

21.0 f 1.4 (-) 17.2 f0.6 (18%) 14.6 Ifr0.8 (30%) 17.1 f 0.4 (17%)

Immunizations were performed i.p. with 5Opg of peptides in the presence of AH. Control mice received adjuvant alone. On days 14 and 21 animals were reinjected with peptides (25 pg) in AH using the same route. Mice were challenged with 50 cercariae 1 day after the injection. Analysis was performed on defined liver sections (n = 3/mouse) sampled at day 42,69 and 96 of infection. The number of eggs present and the collagen and protein content in the section were determined. The protection was expressed as percent reduction of the mean of the result in immunized mice (n = 10) compared to control mice that received only medium (n = 10).

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V. PANCRE

octameric form when compared to mice immunized with the 190-211 peptide (Table 4). DISCUSSION We have recently demonstrated (V. Pancre, in press) that i.p. injection of rSm28GST induced a marked reduction in hepatic lesions in the livers of mice infected by S. ~~~~~~~. In the present work, we tested the ability of i.p. injections of Sm28GST-derived uncoupled peptides to confer protection to S, mansorzi-infected mice. We showed that among the peptides derived from this antigen, the 190-211 peptide, representing the carboxylic end of the molecule, is able to reproduce the protective effect obtained with the whole molecule. In particular, at day 42 of infection, the level of collagen in the liver of 190-211 peptide-immunized mice was close to that observed in normal mice. A significant reduction of splenomegaly was also observed. Moreover, immunized mice seemed durably protected since they survived more than one month after the death of control animals. The absence of any effect after immunization with the other Sm28GST-derived peptides such as 24-43 or 115-l 3 1, previously described as protective, might be explained by the fact that they were used without coupling to a carrier molecule in our study. The 190-211 peptide previously described as predominantly involved in the T-cell response of infected individuals (Auriault et al., 1991) seems to be of particular interest for further study. Xu et al. (1993) have previously demonstrated that immunization of mice with this peptide led to the production of antibodies responsible for the decrease in worm burden and for the reduction of female worm fecundity. Moreover, Grzych et al. (1993) have shown that IgA antibodies of S. mansoni-infected patients from Kenya impaired schistosome fecundity by limiting both the egg laying of mature worms and the hatching capacity of schistosome eggs into viable miracidia. In our system the absence of antibody production against the 190-211 peptide or against rSm28GST in 190-211 peptide-immunized mice might be explained by the fact that peptide antigens are usually not or only weakly immunogenic when used uncoupled. Moreover, with our protocol of immunization with 190-211 peptide, we observed no significant reduction in worm burden that could explain the reduction of hepatic pathology obtained at days 42 and 69 of infection (data not shown). The protection that we observed despite the absence of antibody production suggested a participation of cell dependent mechanisms in the immunity conferred. This was confirmed by the fact that the passive transfer of 190-211 peptide-specific T-cell enriched spleen cells reproduced the protective effect obtained after administration of the 190-211 peptide. Passive transfers of 190-211 peptide-specific T-cell subpopulations are currently under investigation but preliminary results show a low level of protection with CDS+ T-cells whereas CD4+ T-cells not only did not exhibit any effect, but on the contrary, potentiated hepatic lesions (data not shown). In previous work we have demonstrated that in

ef al.

the same experimental conditions of immunization, the administration of Sm28GST protected infected mice and that the protective effect was mediated by cellular processes probably implicating IFN-y (V. Pancrt, in press). We observe here that spleen ceils from 190-211 peptide-immunized mice also produce IFN-y after stimulation with the rSm28GST molecule. In the mouse, the division of CD4+ T-cell clones into IL2 and IFN-y producing Thl and IL4 and IL5 producing Th2 subsets is now well established (Mosmann et a/., 1986). The tendency of Sm28GST and the 190-211 peptide for preferential elicitation of IFN-y and weak IL2 responses with little IL4 production may indicate a role for this antigen in inducing a CD4Th 1 but also CD8 type (Fong and Mosmann, 1990) of T-cell response. Immunization of mice with the 190-211 peptide prior to their infection by S. mansoni allowed an early production of IFN-1, which was not observed in non-immunized controls. In the context of the vaccination of mice with attenuated larval stages, it was shown that vaccinated animals primarily responded by the production of Thl lymphokines and that in contrast to the situation in infected mice, larval schistosome antigens tended to stimulate a stronger Thl-cell response than egg antigens (Lukacs and Boros, 1991). IFN-y, produced by 190-211 peptidespecific cells and by Sm28GST-specific cells, could act either by regulating the antibody response (Pene et al., 1986) or by activating effector cells such as macrophages (James, 1986). Moreover, since IFN-y is a potent inhibitor of collagen synthesis both in civo and in vitro (Czaja et al., 1989) a role for this cytokine can be envisaged in the observed reduction of hepatic pathology. Our protocol of i.p. immunization was initially used to induce rSm28GST-specific CD8+ CTL (V. Pancre, manuscript in preparation). Thus, one possibility could be that we have induced 190-211 peptide-specific CTL acting either directly or through the secretion of cytokines such as IFN-y. In viva induction of CTL by peptide immunization was first described using ovalbumin as the inducing antigen (Carbone and Bevan, 1989). Research in this field has led to the elaboration of potential peptide vaccines against viruses. The problem was to determine the conditions of presentation that gave the most efficient immune response. Some investigators claimed that short synthetic peptides mixed in lipophilic adjuvant are best for priming virus-specific CTL (Gao et al., 1991; Hart et al., 1991; Schulz et al., 1991). Alternatively, the modification of synthetic peptides derived from influenza virus nucleoprotein by linkage to the P3CSS iipopeptide component of E~e~eric~ia coli has been described as a way to elicit CTL responses in vivo (Deres et al., 1989; Schild et al., 1991). We decided to evaluate the protective properties of the 190-211 peptide modified by a lipidic construct, Hda, possessing a simple 14 carbon, linear saturated aliphatic side chain. This lipopeptide was compared to the 190-211 peptide for its capacity to reduce hepatic lesions under the same experimental conditions. We observed that the protective effect of the 190-211 peptide on hepatic lesions was maximal at day 42 of infection but that in the majority of the

Protective effect of Sm28GST-derived C-terminal peptide experiments performed, this effect diminished at day 96 of infection despite the fact that the mice survived. We demonstrate here that the 190-211 lipopeptide is able to induce a stable reduction of the S. munsoni-induced hepatic pathology and long term survival of infected animals. In addition to their ability to induce CTL, Martinon et al. (1992) demonstrated that lipopeptides were also able to induce Th cell stimulation, required both for CTL generation and for antibody production demonstrating that lipopeptides can be successfully used to induce both T- and B-cell responses. We observed no production of IgA or IgG antibodies specific for rSm28GST, the 190-2 11 peptide or the 190-211 lipopeptide after immunization of mice with the 190-211 lipopeptide (data not shown). We are now attempting to establish 190-211 lipopeptide-specific CD8 T-cell lines in order to determine whether these cells might be responsible for the production of IFN-y. The passive transfer of such cells before infection by S. mansoni will be of considerable interest for the understanding of protective mechanisms induced by this peptide. Work using the octameric (MAP 8) construction of the 115-131 fragment of the rSm28GST has demonstrated an increase in the protective effect obtained compared to the monomeric form of the peptide (Wolowczuk et al., 1991). This is why we tested the capacity of the octameric form of the 190-211 peptide to induce an efficient and durable protective response. Surprisingly, we observed no improvement in the reduction of hepatic lesions with this presentation of the 190-211 peptide suggesting that this construct was less able than the lipopeptidic form to induce the correct protective mechanism. In conclusion, this demonstration of an efficient and durable protective effect induced by immunization with a lipopeptidic preparation of a monomeric peptide derived from Sm28GST provides encouragement for the future use of synthetic peptides in the immunoprophylaxis of schistosomiasis. Acknowledgements-This work was supported by INSERM and CNRS and received financial assistance from UNDP/ world bank/WHO Special Program for Research and Training in Tropical Diseases. We thank Dr C. Verwaerde, Dr R. J. Pierce and Dr A. Tsicopoulos for helpful discussions. REFERENCES Auriault C., Wolowczuk I., Gras-Masse H., Marguerite M., Boulanger D., Capron A. and Tartar A. (1991) Epitopic characterization and vaccinal potential of peptides derived from a major antigen of Schistosoma mansoni. Pep. Res. 1, 6-11. Balloul J. M., Grzych J. M., Pierce R. J. and Capron A. (1987a) A purified 28,000 dalton protein from Schistosoma mansoni adult worms protects rats and mice against experimental Schistosomiasis. J. Immun. 138, 3448-3453. Balloul J. M., Sondermeyer P., Dreyer D., Capron M., Grzych J. M., Pierce R. J., Carvallo D., Lecocq J. P. and Capron A. (19876) Molecular cloning of a protective antigen of schistosomes. Nature 326, 149-153. Boros D. L. (1989) Immunopathology of Schistosoma mansoni infection. Clin. Microbial. Rev. 2, 25Ck269.

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Boulanger D., Reid G. D., Sturrock R. F., Wolowczuk I., Balloul J. M., Grezel D., Pierce R. J., Otieno M. F., Guerret S., Grimaud J. A., Butterworth A. E. and Capron A. (1991) Immunization of mice and baboons with the recombinant Sm28GST affects both worm viability and fecundity after experimental infection with Schistosoma mansoni. Parasite immun. 13, 473-490. Carbone F. R. and Bevan M. J. (1989) Induction of ovalbumin-specific cytotoxic T cells by in vivo peptide immunization. J. Exp. Med. 169, 603-612. Castro B., Dormoy R. J., Evin G. and Selve C. (1975) BOP, hexafluorophosphate de benzotriazol N-oxytrisdimethylamino-phosphonium. Tetr. Lett. 14, 1219-1223. Chensue S. W., Wellhausen S. R. and Boros D. L. (1981) Modulation of granulomatous hypersensitivity. II. Participation of Ly 1+ and Ly 2+ T lymphocytes in the suppression of granuloma formation and lymphokine production in Schistosoma mansoni-infected mice. J. Immun. 127, 3630-3635. Courtney M. A., Buchwalder A., Tessier L. H., Jaye M., Benavente A., Balland A., Kilhi V., Lathe R., Tolstoshew P. and Lecocq J. P. (1984) High level production of biologically active human-antitrypsin in Escherischia coli. Proc. natn. Acad. Sci. U.S.A. 81, 669-673. Czaja M. J., Weiner F. R. and Eghbah M. (1987) The differential effects of gamma-interferon on collagen and fibronectine gene expression. J. biol. Chem. 262, 13348-13351. Czaja M. J., Weiner F. R., Takahashi S., Giambrone M. A., Van Der Meide P., Schellekens H., Biempica L. and Zern M. A. (1989). y-interferon treatment inhibits collagen deposition in murine Schistosomiasis. Hepatology 5, 795-800. Deres K., Schild H., Wiesmiiller K. H., Yung G. and Rammensee H. G. (1989) In vivo priming of virus-specific cytotoxic T lymphocytes with synthetic lipopeptide vaccine. Nature 342, 561-564. Fidel P. L. and Boros D. L. (1990) Regulation of granulomatous inflammation in murine Schistosomiasis. IV. Antigen-induced suppressor T cells down-regulate proliferation and IL-2 production. J. Immun. 145, 1257-1264. Fong T. A. T. and Mosmann T. R. (1990) Alloreactive murine CD8+ T cell clones secrete the Thl pattern of cytokines. J. Immun. 144, 1744-1752. Gao X. M., Zheng B., Liew F. Y., Brett S. and Tite J. (1991) Priming of influenza virus-specific cytotoxic T lymphocytes in vivo by short synthetic peptides. J. Immun. 147, 3268-3273. Grzych J. M., Pierce E., Cheever A., Caulada Z. A., Caspar P., Henry S., Lewis F. and Sher A. (1991) Egg deposition is the major stimulus for the production of Th2 cytokines in murine Schistosomiasis mansoni. J. Immun. 146, 1322-l 327. Grzych J. M., Grezel D., Xu C. B., Neyrinck J. L., Capron M., Ouma J. H., Butterworth A. E. and Capron A. (1993) IgA antibodies to a protective antigen in human Schistosomiasis mansoni. J. Immun. 150, 527-535. Hart M. K., Weinhold K. J., Scearce R. M., Washburn E. M., Clark C. A., Palker T. J. and Haynes B. F. (1991) Priming of anti-human carrier-free immunodeficiency virus (HIV) CD8+ cytotoxic T cells in vivo by HIV synthetic peptides. Proc. natn. Acad. Sci. U.S.A. 88, 9448-9452. James S. L. (1986) Activated macrophages as effector cells of protective immunity to Schistosomiasis. Immunol. Res. 5, 13946.

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Lopez de Leon A. and Rojkind M. (1985) A simple method for collagen and total protein determination in formalin-fixed paraffin-embedded sections, J. Histochem. Cytochem. 33, 137-743. Lu Y. P., Clavijo P., Galantino M., Shen Z., Liu W. and Tam J. P. (1991) Chemically unambiguous peptide immunogen: Preparation, orientation and antigenicity of purified peptide conjugated to the multiple antigen peptide system. Mofec. Immun. 28, 623-630. Lukacs N. W. and Boros D. L. (1991) Identification of larval cross-reactive and egg-specific antigens involved in circumoval granuloma formation in murine Schistosomiasis mansoni. Infect. Immun. 59, 3231-3242. Martinon F., Gras-Masse H., Boutillon C., Chirat F., Deprez B., Guillet J. G., Gomard E., Tartar A. and Levy J. P. (1992) Immunization of mice with lipopeptides bypasses the prerequisite for adjuvant. J. Immun. 149, 34163422. Mathew R. C. and Boros D. L. (1986) Anti L3T4 antibody treatment suppresses hepatic granuloma formation and abrogates antigen-induced interleukin 2 production in Schistosoma mansoni infection. Infect. Immun. 54, 820-826. Mathew R. C., Ragheb S. and Boros D. L. (1990) Recombinant IL-2 therapy reverses diminished granulomatous responsiveness in anti L3T4-treated, Schistosoma mansoniinfected mice. J. Zmmun. 144, 43564361. Merrifield R. B. (1983) Solid phase peptide synthesis. J. Am. Chem. Sot. 85, 2149-2154. Mosmann T. R., Cherwinski H., Bond M. W., Giedlin M. A. and Coffman R.L. (1986) Two types of murine helper T cell clones. I. Definition according to profiles of lymphokine activities and secreted proteins. J. Immun. 136, 2348-2357. Pearce E. J., Caspar P., Grzych J. M., Lewis F. A. and Sher A. (1991) Down-regulation of Thl cytokine production accompanies induction of Th2 responses by a parasitic helminth, Schistosoma mansoni. J. exp. Med. 173, 159-166. Pene J., Rousset F., Briere F., Chretien I., Bonnefoy J. Y., Spits H., Yokota T., Arai N., Bancherau J. and Deries J. (1986) IgE production by normal human lymphocytes is induced by interleukin 4 and suppressed by interferons y and t( and prostaglandin E2. Proc. natn. Acad. Sci. U.S.A. 85, 6880-6884. Perrin P. J. and Phillips S. M. (1988) The molecular basis of granuloma formation in Schistosomiasis. I. A T cell-derived suppressor effector factor. J. Immun. 141, 17141719.

et al.

Phillips S. M and Lammie P. (1986) Immunopathology of granuloma formation and fibrosis in Schistosomiasis. Parasitol. Today 2, 296302. Schild H., Deres K., Wiesmiiller K. H., Jung G. and Rammensee H. G. (1991) Efficiency of peptides and lipopeptides for in vivo priming of virus-specific cytotoxic T cells. Eur. J. Immun. 21, 2649-2654. Schulz M., Zinkernagel K. and Hengartner H. (1991) Peptideinduced antiviral protection by cytotoxic T cells. Proc. natn. Acad. Sci. U.S.A. 88, 991-995. Tam J. P. (1988) Synthetic peptide vaccine design: Synthesis and properties of a high-density multiple antigen peptide system. Proc. natn. Acad. Sci. U.S.A. 85, 5409-5413. Taylor M. G., Vidal A., Torpier G., Meyer D. J., Roitsch C., Balloul J. M., Southan C., Sondermeyer P., Premble S., Lecocq J. P., Capron A. and Ketterer B. (1988) The glutathione transferase activity and tissue distribution of a cloned Mr 28 K protective antigen of Schistosoma mansoni. Eur. Molec. Biol. Org. J. 7, 4655472. Wolowczuk I., Auriault C., Gras-Masse H., Vendeville C., Balloul J. M., Tartar A. and Capron A. (1989) Protective immunity in mice vaccinated with the Schistosoma mansoni P-28-l antigen. J. Immun. 142, 1342-1350. Wolowczuk I., Auriault C., Bossus M., Boulanger D., Gras-Masse H., Mazingue C., Pierce R. J., Grezel D., Reid G. D., Tartar A. and Capron A. (1991) Antigenicity and immunogenicity of a multiple peptidic construction of the Schistosoma mansoni Sm28GST antigen in rat, mouse and monkey. J. Immun. 146, 1987-1995. Wolowczuk I., Kimani G., Boulanger D., Butterworth A. E., Gras-Masse H., Tartar A., Ouma J. H., Tarara R., Reid G. D., Koech D., Auriault C. and Capron A. (1994) A study of human and primate lymphocyte responses to synthetic peptides of the Sm28GST protective antigen of Schistosoma mansoni. J. Clin. Microbial. (in press). Wyler D. J., Ehrlich H. P., Postlethwaite A. E., Raghow R. and Murphy M. M. (1987) Fibroblast stimulation in Schistosomiasis. VII. Egg granulomas secrete factors that stimulate collagen and fibronectin synthesis. J. Immun. 138, 1581-1586. Xu C. B., Verwaerde C., Gras-Masse H., Fontaine J., Bossus M., Trottein F., Wolowczuk I., Tartar A. and Capron A. (1993) Schistosoma mansoni 28-kDa glutathione Stransferase and immunity against parasite fecundity and egg viability. J. Immun. 150, 940-949.